FCH 532 Lecture 4a
Chapter 5: Molecular biology overview
Transcription
• Catalyzed by RNA polymerase.
• Couples NTPs (ATP, CTP, GTP, UTP) to make RNA
• (RNA)n residues + NTP  (RNA)n+1 residues + P2O74• 5’  3’ nucleotides are added to the free 3’-OH group
• Nucleotides must meet Watson-Crick base pairing
requirements with the template strand
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Figure 5-23 Action of RNA polymerases.
Transcription
• Transcribes only one template DNA strand at a time.
• RNA polymerase will move along the duplex DNA it is
transcribing and creates a transcription bubble
• This forms a short DNA-RNA hybrid with newly
synthesized RNA.
• DNA template strand is read 3’  5’
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Figure 5-24 Function of the transcription bubble.
Transcription
• DNA template contains control sites consisting of specific base
sequences that specify where the RNA polymerse initiates
transcription and the rate of transcription.
• activators and repressors control the sites in prokaryotes.
• Transcription factors bind to these sites in eukaryotes.
• messenger RNA (mRNA) - RNAs that encode proteins
• Rates at which cells synthesize a protein are determined
by the rate at which mRNA synthesis is initiated.
• Promoter-in prokaryotes-a sequence that precedes the
transcriptional initiation site.
Transcription
• Prokaryotes can control transcriptional initiation in complex
manners.
• Example E. coli lac operon.
• Has 3 consecutive genes (Z, Y, and A) that are necessary to
metabolize lactose.
• In the absence of lactose, the lac repressor protein binds a
control site in the lac operon called an operator.
• This prevents the RNA polymerase from initiating transcription.
• If lactose is present, some of the lactose is converted to
allolactose which binds to the lac repressor causing it to fall of
the operator sequence.
• This allows RNA polymerase to initiate transcription of the
genes.
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Figure 5-25 Control of transcription of the lac operon.
Eukaryotic RNA undergoes posttranscriptional modification
• In order for mRNAs in eukaryotes to become functional, they
must undergo modifications.
• 7-methylguanosine-containing “cap” is added to the 5’ end.
•  250 nucleotide polyadenylic acid [poly(A)] tail is added to
the 3’ end.
• Undergo gene splicing in which RNA segments called introns
are excised from the RNA and the remaining exons are
rejoined to form the mature mRNA.
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Figure 5-26 Post-transcriptional processing of
eukaryotic mRNAs.
mRNA
• In prokaryotes, transcription and translation both
take place in the cytosol.
• Prokaryotic mRNAs have a short lifetime (avg. 1-3
min). They are degraded by nucleases.
• Rapid turnover in prokaryotes allows the prokaryote
to respond quickly to the environment.
• In eukaryotic cells, RNAs are transcribed and posttranslationally modified in the nucleus, then sent to
cytosol.
• Eukaryotic mRNAs have lifetimes of several days.
Translation: Protein synthesis
• Polypeptides are synthesized from mRNA by ribosomes.
• Ribosomes are 2/3 rRNA (ribosomal RNA) and 1/3 protein.
• Prokaryote ribosomes approx. 2500 kD, eukaryotes 4300 kD
• Transfer RNAs (tRNAs) deliver amino acids to the ribosome.
• mRNA sequences can be broken down to codons-consecutive
3-nucleotide segments that specify a particular amino acid.
• Once the mRNA binds to the ribosome, they specifically bind
to the tRNA that is covalently linked to an amino acid.
Figure 5-27 Transfer RNA (tRNA) drawn in its
“cloverleaf” form.
tRNA has 76 nucleotides
Has an anticodoncomplementary sequence
to the mRNA sequence
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Amino acid is linked to the
3’ end of the tRNA to form
aminoacyl-tRNA.
tRNAs are “charged” with
amino acids by specific
enzymes (aminoacyltRNA synthetases or
aaRSs)
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Figure 5-28 Schematic diagram of translation.
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Figure 5-29 The ribosomal reaction forming a peptide
bond.
Genetic code
•
•
•
•
•
•
•
•
•
Correspondence between the sequence of bases in a codon and the
amino acid residue it specifies.
Nearly universal.
4 possible bases (U[T], C, A, and G) can occupy three positions of
codon, therefore 43 = 64 possible codons.
61 codons specify amino acids, and three UAA, UAG, and UGA are
stop codons (cause ribosome to end polypeptide synthesis and
release the transcript).
All but two amino acids (Met, Trp) are specified by more than one
codon.
Three (Leu, Ser, Arg) are specified by six codons.
Synonyms-multiple codons can code the same amino acid.
tRNA may recognize up to 3 synonymous codons because the 5’
base of a codon and 3’ base of the anticodon can interact in ways
other than via Watson-Crick base pairs.
Translation is initiated at the AUG codon (Met) but this tRNA differs
from the tRNA for internal amino acid the Met codon.
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Figure 5-30 Nucleotide reading frames.
DNA replication
• DNA is replicated similar to RNA with some differences:
• 1. Deoxynucleotide triphosphates (dNTPs) are used
instead of NTPs
• 2. Enzyme is the DNA polymerase
• Other differences:
• RNA polymerase can link together two nucleotides on
DNA template, but DNA polymerase can only extend (in
the 5’ to 3’) direction an existing polynucleotide that is
base paired to the template strand.
• DNA polymerase needs an oligonucleotide primer to
initiate synthesis.
• Primers are RNA.
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Figure 5-31 Action of DNA polymerases.
DNA strands replicated in different
ways
• DNA strands are simultaneously replicated.
• Takes place at replication fork - junction where the two
parental DNA are pried apart and where the two
daughter strands are synthesized.
• Leading strand is continuously copied from the 3’ to 5’
parental template in the 5’ to 3’ direction
• Lagging strand is discontinuously replicated in pieces
from the 5’ to 3’ parental strands.
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Figure 5-32a Replication of duplex DNA in E. coli.
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Figure 5-32bReplication of duplex DNA in E. coli.
DNA strands replicated in different
ways
• DNA strands are simultaneously replicated.
• Takes place at replication fork - junction where the two
parental DNA are pried apart and where the two
daughter strands are synthesized.
• Leading strand is continuously copied from the 3’ to 5’
parental template in the 5’ to 3’ direction
• Lagging strand is discontinuously replicated in pieces
from the 5’ to 3’ parental strands.
• E. coli has 2 DNA polymerases necessary for survival.
DNA polymerase III (Pol III) synthesizes the leading
strand and most of the lagging strand.
• DNA polymerase I (Pol I) removes RNA primers and
replaces them with DNA. This enzymes also has a 5’ to
3’ exonuclease activity.
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Figure 5-33 The 5¢ ® 3¢ exonuclease function of DNA
polymerase I.
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Figure 5-34 Replacement of RNA primers by DNA in
lagging strand synthesis.
Lagging strand synthesis
• Synthesis of the leading strand of DNA is completed by
the replacement of the RNA primer by DNA.
• Lagging strand is completed after nicks between multiple
disconinuously synthesized segments are sealed by
DNA ligase.
• Catalyzes the links of 3’-OH to 5’-phosphate groups.
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Figure 5-35 Function of DNA ligase.
Errors in DNA sequence can be
corrected
• RNA polymerase has an error rate of 1 in 104 base pairs
in E. coli.
• Pol I and Pol III have 3’  5’ exonuclease activities.
• This activity degrades the newly synthesized 3’ end of a
daughter strand one nucleotide at a time to edit out
mistakes that are sometimes incorporated.
• Other enzymes are present that detect and correct errors
in DNA damage that occurs from UV radiation and
mutagens (chemical substances that damage DNA) and
hydrolysis.
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Figure 5-36 The 3¢ ® 5¢ exonuclease function of DNA
polymerase I and DNA polymerase III.
Molecular cloning
• Clone-a collection of identical organisms that are derived
from a single ancestor.
• Molecular cloning techniques - genetic engineering,
recombinant DNA technology.
• Main idea is to insert a DNA segment of interest into an
automously replicating cloning vector so that the DNA
segment is replicated with the vector.
• Cloning into a chimeric vector in a suitable host
organism results in large amounts of the inserted DNA
segment.
Restriction endonucleases
• Restriction enzymes (endonucleases) cleave DNA at
specific sequences within a polynucleotide.
• Bacteria use restriction/modification systems as a small
scale immune system for protection from infection by
foreign DNA.
• W. Arber and S. Linn back in 1969 showed that plating
efficiencies of bacteriophage lambda grown in E. coli
strains C, K-12, and B were very different.
E . coli strain on which parental
phage had been grown
C
K-12
B
E . coli strain for plating phage
C
1
1
1
K-12
<10-4
1
<10-4
B
<10-4
<10-4
1
* The DNA of phage which had been grown on strains K-12 and
B were found to have chemically modified bases which were
methylated.
* Additional studies with other strains indicate that different
strains had specific methylated bases.
* Typical sites of methylation include the N6 position of
adenine, the N4 position of cytosine, or the C5 position of
cytosine.
* In addition, only a fractional percentage of bases were
methylated (i.e. not every adenine was methylated, for
example) and these occurred at very specific sites in the
DNA.
* A characteristic feature of the sites of methylation, was
that they involved palindromic DNA sequences.
* In addition to possessing a particular methylase, individual
bacterial strains also contained accompanying specific
endonuclease activities.
* The endonucleases cleaved at or near the methylation
recognition site.
* These specific nucleases, however, would not cleave at
these specific palindromic sequences if the DNA was
methylated.
Thus, this combination of a specific methylase and
endonuclease functioned as a type of immune system for
individual bacterial strains, protecting them from infection by
foreign DNA (e.g. viruses).
* In the bacterial strain EcoR1, the sequence GAATTC
will be methylated at the internal adenine base (by the
EcoR1 methylase).
* The EcoR1 endonuclease within the same bacteria will
not cleave the methylated DNA.
* Foreign viral DNA, which is not methylated at the
sequence "GAATTC" will therefore be recognized as
"foreign" DNA and will be cleaved by the EcoR1
endonuclease.
* Cleavage of the viral DNA renders it non-functional.
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Figure 5-37 Restriction sites.
Types of restriction
endonucleases
• Type I and Type II restriction enzymes have
both endonuclease and methylase activity on
the same polypeptide.
• Type I enzymes cleave the DNA at a location of
at least 1000 bp away from the recognition
sequence.
• Type III enzymes cleave DNA from 24 to 26 bp
away from the recognition site.
• Type II restriction enzymes are separate from
their methylases. These enzymes cleave DNAs
at specific sites within the recognition sequence
(table 5-4).
Restriction endonucleases
recognize pallindromic sequences
• Palindrome-a word, verse, or sentence that reads the
same forwards or backwards.
• Restrction enzymes cleave 2 DNA strands at positions
that are symmetrically staggered about the center of the
palindromic sequence.
• Yields restction fragments with complementary singlestranded ends (1-4 nt in length called sticky ends).
• The sticky ends can associate by complementary base
pairing with other fragments generated by the same
restriction enzyme.
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Figure 5-37 Restriction sites.
Restriction maps
• After digest with DNA restriction endonuclease the fragments can
be separated according to size by gel electrophoresis.
• DNA can be separated according to size by agarose or
polyacrylamide.
• Duplex DNA is detected by staining with intercalating, planar,
aromatic cations such as ethidium, acridine orange, or
proflavin, between stacked base pairs. New stains like SYBR
are available that are notThese exhibit flurorescence under UV
light.
• As little as 50 ng of DNA may be detected in a gel by staining it
with ethidum bromide.
• Can also be used to visualize single stranded DNA or RNA.
• Can be used to generate a restriction map.
Figure 5-38 Agarose gel electrophoretogram of
restriction digests.
Digest of Agrobacterium
radiobacter plasmid pAgK84
digested with:
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A. BamHI, B. PstI, C. BglII, D.
HaeIII, E. HincII, F. SacI, G.
XbaI, H. HpaI. Lane I contains l
phage DNA digested with
HindIII as standards.
23130 bp
9416 bp
6557 bp
4361 bp
2322 bp
2027 bp
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Figure 5-39 Construction of a restriction map.
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Figure 5-40 Restriction map for the 5243-bp circular
DNA of SV40.